1. Field of the Invention
The present invention relates to a recombinant expression vector using phosphoribulokinase as a fusion partner and a process for preparing human parathyroid hormone therewith, more specifically, to a recombinant expression vector which is prepared by inserting a human parathyroid hormone gene containing an urokinase-specific cleavage site into a L-arabinose inducible vector containing a phosphoribulokinase gene fragment of Rhodobacter sphaeroides or its mutated gene- as a fusion partner, a recombinant microorganism transformed with the said expression vector, and a process for preparing human parathyroid hormone on a large scale by cultivating the said microorganism in a medium containing L-arabinose.
2. Description of the Prior Art
Osteoporosis is a disease causing harmful effects such as fracture even by small impact which results from reduction in mass of bone compared with normal people and weakness of bone tissue. The advance of medical science and biology leads to continuous increase in population of old age, which results in continuous increase of patients suffering from osteoporosis. Therefore, osteoporosis becomes a big social problem at present when number of old people living alone increases gradually according to a tendency of a nuclear family.
In general, in a normal bone tissue, balance between activities of osteoclast, a bone-destructing cell and osteoblast, a bone-forming cell is accomplished, which results in constant remodeling of bone tissue. In a normal body, osteoclast surpasses osteoblast in functioning according to increase in age, which results in overall decrease in bone density. In a patient suffering from osteoporosis, such a disruption in balance between activities of osteoclast and osteoblast is much higher than in normal case.
Although the cause of disruption in balance between activities of osteoclast and osteoblast has not been known clearly, it has been found that reduction in secretion of estrogen, a female hormone after the menopause causes osteoporosis type 1 which suffers women after the menopause largely. Thus, estrogen has been administered for the treatment of osteoporosis type 1, and many patients are, however, reluctant to use estrogen because of side effects such as high probability of attack of breast cancer, endometrium cancer, etc. Also, estrogen cannot be used for the treatment of osteoporosis type 2 which has been known to be induced by a cause different from that inducing osteoporosis type 1.
Calcitonin which inhibits activities of osteoclast to suppress resorption of bone tissue has been used as an agent which compensates for shortcomings of estrogen, an agent for the treatment of osteoporosis type 1 and treats osteoporosis type 2 not to be cured by estrogen. However, estrogen and calcitonin have no effect on increase in mass of bone which is already lost and only prevent further decrease in bone density. Therefore, they are improper for the effective treatment of osteoporosis.
Recently, parathyroid hormone (PTH) has been noticed as a good agent for the treatment of osteoporosis since PTH has an effect of increasing bone density as well as an effect of preventing reduction in bone density and its side effects have not been reported. Preproparathyroid hormone (preproPTH) which consists of 115 amino acids and is produced in main cells of parathyroid gland is processed and transformed into proPTH consisting of 92 amino acids while traveling through endoplasmic reticulum. Then, proPTH is further processed and transformed into mature PTH consisting of 84 amino acids while traveling through Golgi apparatus. PTH synthesized by the said processes is secreted into blood and transported to target organs, i.e., bone and kidney. Secreted PTH has a half-life of only 18 minutes.
PTH activates Ca2+ pump in bone cell membrane to promote CaHPO4 mobilization from bone which results in increase in blood Ca2+ level within several minutes. Moreover, when PTH is secreted continuously, it activates osteoclasts already existed, stimulates formation of new osteoclasts, and inhibits activities of osteoblasts temporarily, which results in inhibition of Ca2+ deposition into bone and stimulation of Ca2+ release to increase secretion of Ca2+ and PO43xe2x88x92 into blood. On the other hand, secretion of PTH is regulated by blood Ca2+ concentration through strong feedback mechanism. That is, 10% reduction-of blood Ca2+ concentration in a short time doubles secretion of PTH. When blood Ca2+ concentration is low for a long time, even 1% reduction of blood Ca2+ . concentration doubles secretion of PTH.
Unlike such a regulatory function of PTH in a living body, it has been reported that PTH stimulates formation of bone when external PTH is administered in a small dose intermittently (see: Tam, C. S. et al., Endocrinology, 110:506-512(1982)). The use of PTH for the treatment of osteoporosis is based on the said stimulatory function of PTH in formation of bone. Although the mechanism of stimulatory function of PTH in formation of bone has not been clearly understood, hypotheses such as inhibition of PTH secretion by the administered PTH, direct stimulation of osteoblasts and indirect stimulation of formation of bone through growth factor including insulin-like growth factor-1 (IGF-1) and transforming growth factor-xcex2 (TGF-xcex2) have been suggested.
In order to treat osteoporosis by using PTH, administration of PTH for a long time is essentially required. However, processes for mass production of PTH have not been established so far, and practical application of PTH for the treatment of osteoporosis has been in a difficult situation. Thus, the present inventors have studied mass production of PTH from a recombinant microorganism employing genetic engineering technology and made an effort to remove amino-terminal methionine residue during expression of PTH in E. coli since Ser-Val-Ser amino acid sequence at amino-terminus of PTH has been reported to be essential for biological activity of PTH.
Methionine-specific amino peptidase, an enzyme removing translation-initiating methionine at amino-terminus of expressed proteins exists in E. coli which is widely used as a host cell for expression of a recombinant protein. However, when foreign proteins are expressed in large quantities in E. coli, removal of amino-terminal methionine is not achieved occasionally. Such a phenomenon has to be solved to construct an expression system of a protein whose amino acid sequence at amino-terminus affects its own biological activity, e.g., PTH.
In order to solve the said problem, three methods may be used mainly as followings: First, a desired protein is secreted into periplasm of E. coli or cultured medium in an amino-terminus processed form by expressing the desired protein in a fused form with secretion signal sequence at amino-terminus. The said method has an advantage that a mature protein is obtained by intracellular activity, while it has a shortcoming that yield of expression is relatively low. Secondly, after only a desired protein is expressed in E. coli and isolated from E. coli in a methionine-attached form at amino-terminus, it is digested with amino peptidase to obtain a mature protein. The said method has a shortcoming that purification of the protein is complex since separation of amino-terminal methionine-removed proteins from methionine-attached proteins is difficult. Thirdly, after a fusion protein where a desired protein is fused with another protein is expressed in E. coli and isolated, the fused partner is removed from the fusion protein employing an enzyme or a chemical agent to obtain a mature desired protein. The said method has an advantage of high efficiency of expression of a desired protein as well as production of an amino-terminal methionine-free protein.
On the other hand, methods for obtaining a desired protein from a fusion protein are largely classified into cleaving methods employing chemicals and enzymes. Among them, the cleaving methods employing chemicals have an advantage of low cost because of an use of chemicals of low-price. However, they have a shortcoming that an additional step for purifying a desired protein from byproducts is required since use of chemicals gives rise to produce various byproducts as well as a desired protein due to low specificity of chemicals to a cleaved site. On the contrary, the problem of the cleaving methods employing chemicals can be solved by the usage of enzymes due to their high specificity to a cleaved site. However, the cleaving methods employing enzymes have difficulties in practical application on industrial scale since price of enzymes is high.
In this regard, studies on the economical use of enzymes cleaving a fusion protein have been carried out in the art. However, mass production of Factor Xa, thrombin, enterokinase, etc. which are enzymes used for the said purpose is rather limited. Thus, the cleaving methods employing enzymes have not been used widely on an industrial scale regardless of their various advantages. Accordingly, there are strong reasons for exploring a third enzyme which can be produced in large quantities economically to be used efficiently for the cleaving methods employing enzymes. Since mass production of urokinase (two chain urokinase type plasminogen activator), a serine protease used as a thrombus-dissolving agent have been already developed and active urokinase can be prepared in large quantities employing expression system of prokaryote such as E. coli (see: W. E. Holmes et al, Bio/Technology, 3:923-929 (1985)), urokinase can be produced and obtained in large quantities economically compared with other enzymes such as Factor Xa, etc. Naturally, urokinase has been proposed as a potential candidate for the economical cleavage of a fusion protein and isolation of a desired protein.
Under the circumstances, the present inventors have determined amino acid sequence of urokinase-specific cleavage site within a protein, and discovered that cleavage efficiency is high when an amino acid sequence of -X-Gly-Arg (wherein, X represents Pro, Thr, Ile, Phe or Leu), an urokinase-specific cleavage site is present between a desired protein and a fusion partner in a fusion protein. Also, they have discovered that the highest cleavage efficiency cab be obtained in the presence of -Thr-Gly-Arg among the sequences (see: Korean patent laid-open publication No. 97-6495).
The present inventors have made an effort to prepare amino-terminal methionine-free PTH from a recombinant E. coli in large quantities, and discovered that recombinant PTH having an activity of native human PTH can be prepared on a large scale, by a process which comprises the steps of: inserting a human PTH gene which contains a urokinase-specific cleavage site and uses universal codon of E. coli into a L-arabinose inducible vector containing a phosphoribulokinase (xe2x80x9cPRKxe2x80x9d) gene fragment of Rhodobacter sphaeroides or its mutated gene as a fusion partner to construct an expression vector, transforming E. coli with the said expression vector, isolating a fusion protein from the said transformed cell, and cleaving the fusion protein with urokinase.
A primary object of the invention is, therefore, to provide a recombinant expression vector which is prepared by inserting a human PTH gene containing an urokinase-specific cleavage site into a L-arabinose inducible vector containing a PRK gene fragment or its mutated gene.
The other object of the invention is to provide a recombinant microorganism transformed with the said expression vector.
Another object of the invention is to provide a process for preparing human PTH on a large scale by cultivating the said microorganism in a medium containing L-arabinose.